Purified human prostate antigen

ABSTRACT

A prostate antigen distinct from prostatic acid phosphatase has been detected in normal, benign hypertrophic and malignant prostatic tissues, but not in other human tissues. The prostate antigen was purified to homogeneity from prostatic tissues by ammonium sulfate precipitation, DEAE-BioGel A anion exchange chromatography, molecular sievings on Sephadex G-100 and Sephadex G-75, and preparative polyacrylamide gel electrophoresis. The purified prostate antigen shows a single protein band on analytical polyacrylamide gel electrophoresis and isoelectric focusing. The molecular weight of purified antigen was estimated by Sephadex G-75 gel filtration to be 33,000 and by sodium dodecyl sulfate polyacrylamide gel electrophoresis to be 34,000 with no subunit. The prostate antigen had an isoelectric point of 6.9.

This invention was supported in part by Grants No. CA-15126 and CA-15437from the National Cancer Institute, U.S. Public Health Service.

DESCRIPTION OF THE INVENTION

This application is a continuation-in-part of copending, commonlyassigned U.S. patent application Ser. No. 108,217 now abandoned, filedDec. 28, 1979, the contents of which are incorporated by referenceherein.

TECHNICAL FIELD OF THE INVENTION

This invention relates to a diagnostic reagent and method for theimmunochemical detection of a human prostate antigen which is distinctfrom prostatic acid phosphatase. More particularly, this inventionrelates to a novel purified human prostate antigen and antibodiesspecific thereto which are suitable for use in prostatic cancerdetection by laboratory methods.

BACKGROUND ART

Prostate cancer is very prevalent in old age, with approximately onehalf of all males over age 70 having been shown to develop prostaticcancer. This high incidence of prostate malignancy has led to the searchfor markers which may be used for its detection. The elevation of serumacid phosphatase activity in patients having metastasized prostatecarcinoma was first reported by Gutman et al. in J. Clin. Invest. 17:473 (1938). In cancer of the prostate, prostatic acid phosphatase isreleased from the cancer tissue into the blood stream with the resultthat the total serum acid phosphatase level greatly increases abovenormal values. Numerous studies of this enzyme and its relation toprostatic cancer have been made since that time, e.g., see the review byYam in Amer. J. Med. 56: 604 (1974). However, the measurement of serumacid phosphatase by conventional spectrophotometric methods often failsto detect prostatic cancer in its early stages. In general, the activityof serum acid phosphatase is elevated in about 65-90 percent of patientshaving carcinoma of the prostate with bone metastatis; in about 30percent of patients without roentgenological evidence of bonemetastasis; and in about only 5-10 percent of patients lackingclinically demonstrable metastasis.

Prior art attempts to develop a specific test for prostatic acidphosphatase have met with only limited success because techniques whichrely on enzyme activity on a so-called "specific" substrate cannot takeinto account other biochemical and immunochemical differences among themany acid phosphatases which are unrelated to enzyme activity ofprostate origin. In the case of isoenzymes, i.e. genetically definedenzymes having the same characteristic enzyme activity and a similarmolecular structure but differing in amino acid sequences and/or contentand therefore immunochemically distinguishable, it would appearinherently impossible to distinguish different isoenzyme forms merely bythe choice of a particular substrate. It is therefore not surprisingthat none of these prior art methods is highly specific for the directdetermination of prostatic acid phosphatase activity; e.g. see Cancer 5:236 (1952); J. Lab. Clin. Med. 82: 486 (1973); Clin. Chem. Acta. 44: 21(1973); and J. Physiol. Chem. 356: 1775 (1975).

In addition to the aforementioned problems of non-specificity whichappear to be inherent in many of the prior art reagents employed for thedetection of prostate acid phosphatase, there have been reports ofelevated serum acid phosphatase associated with other diseases, whichfurther complicates the problem of obtaining an accurate clinicaldiagnosis of prostatic cancer. For example, Tuchman et al. in Am. J.Med. 27: 959 (1959) have noted that serum acid phosphatase levels appearto be elevated in patients with Gaucher's disease.

Due to the inherent difficulties in developing a "specific" substratefor prostrate acid phosphatase, several researchers have developedimmunochemical methods for the detection of prostate acid phosphatase.However, the previously reported immunochemical methods have drawbacksof their own which have precluded their widespread acceptance. Forexample, Shulman et al., in Immunology 93: 474 (1964) described animmunodiffusion test for the detection of human prostate acidphosphatase. Using antisera prepared from a prostatic fluid antigenobtained by rectal massage from patients with prostatic disease, nocross-reactivity precipitin line was observed in the double diffusiontechnique against extracts of normal kidney, testicle, liver and lung.However, this method has the disadvantages of limited sensitivity, evenwith the large amounts of antigen employed, and of employing antiserawhich may cross-react with other, antigenically unrelated serum proteincomponents present in prostatic fluid.

Chu et al. in International Patent Application Publication No. WO79/00475, the contents of which are incorporated by reference herein,describe a new method for the detection of prostatic acid phosphataseisoenzyme patterns associated with prostatic cancer which obviates manyof the above drawbacks. However, practical problems are posed by theneed for a source of cancerous prostate tissue from which thediagnostically relevant prostatic acid phosphatase isoenzyme patternsassociated with prostatic cancer are extracted for the preparation ofantibodies thereto.

In recent years considerable effort has been spent to identify enzyme orantigen markers for various types of malignancies with the view towardsdeveloping specific diagnostic reagents. The ideal tumor marker wouldexhibit, among other characteristics, tissue or cell-type specificity,and would be released into the circulation or other biological milieuwhich is easily obtained from individuals. Previous investigators havedemonstrated the occurrence of human prostate tissue-specific antigens.

For example, R. H. Flocks et al., I. J. Urol. 84: 134 (1960) immunizedrabbits with an extract of BPH (benign prostatic hypertrophy) prostatetissue and showed the presence of tissue specific antiprostaticantibodies by a gel diffusion technique. However, they presented no dataat all to indicate the nature of the reactive antigen. The precipitationlines formed by antiserum and prostate extract appear due to thereaction of prostatic acid phosphatase and its antibodies.

R. J. Ablin et al., J. Immunol. 104: 1329 (1970) and R. J. Ablin, Cancer29: 1570 (1972) have also demonstrated the occurrence of human prostatetissue-specific antigens. By using antiserum obtained from immunizingrabbits with extracts of normal prostate, Ablin et al. showed twoantigenic components in human prostate. One of these was identified asprostatic acid phosphatase, while the specificity of other was shown tobe a non-prostatic tissue antigen. The xenoantibodies reactive to thesecond antigen could not be abolished by treating the antiserum withhuman prostatic fluid. Furthermore, this antigen was shown to bedeficient in benign and malignant prostatic tissues. Incontradistinction, the prostate antigen of the present invention ispresent in all prostate tissue, (normal, benign or malignant) in almostequal amounts. Further, it is detectable in prostatic fluid and culturedhuman prostatic malignant cells and its medium as well. The Ablinarticles describe absorption of antisera to the antigens describedtherein with prostatic fluid, after which a precipitin line was stilldetected. Absorption of antibodies against the present antigen withprostatic fluid gives no precipitin line, indicating that the presentantigen is present in prostatic fluid while that of Ablin et al. is not.

C. W. Moncure et al., Cancer Chemother. Rep. 59: 105, (1975) alsodemonstrated the occurrence of a human prostate tissue-specific antigenpreparation which does not bind to DEAE sepharose at pH 8.0, as does theantigen of the present invention. This characteristic most probablyindicates a significant structural difference between the presentprotein and that of Moncure et al.

Thus, there is still a need for simple, reliable, sensitive and specificreagents and techniques to detect prostatic cancer with acceptablediagnostic accuracy and without the aforementioned difficulties of theprior art. The present invention fills such needs.

DISCLOSURE OF THE INVENTION

It is a general object of the present invention to provide a purifiedhuman prostate antigen useful in preparing an improved diagnosticreagent suitable for the immunochemically specific detection ofcirculating human prostate antigen in blood, urine or other body fluids.

Another object of the present invention is to provide rapid and simple,yet highly specific and sensitive, immunochemical techniques andreagents useful in the early detection of prostatic cancer.

A further object of this invention is to provide a new marker formonitoring prostatic cancer and the effectiveness of curative therapytherefor.

An additional object of this invention is to provide useful monoclonalantibodies to human prostate antigen.

Upon study of the specification and appended claims, further objects,features and advantages of the present invention will become more fullyapparent to those skilled in the art to which this invention pertains.

BEST MODE FOR CARRYING OUT THE INVENTION

Briefly, the above and other objects, features and advantages of thepresent invention are attained in one aspect thereof by providing apurified human prostate antigen which is distinct from prostatic acidphosphatase.

In a second aspect, the present invention provides antisera which arehighly specific to a purified human prostate antigen and which do notimmunochemically cross-react with prostatic acid phosphatases or withacid phosphatases originating from other tissues.

In a third aspect of the present invention, there is provided animmunochemical method for the detection of prostatic cancer whichexhibits high sensitivity, good specificity and substantially no falsepositive results for tumor other than the prostate.

In a fourth aspect, the present invention provides specific markerantibodies against human prostate antigen which are useful, e.g., ascarriers for in vivo radioimmunodetection of prostate cancer andimmunospecific chemotherapy of prostatic cancer.

According to the present invention, antigenic preparations from eithernormal or cancerous human prostate tissue, prostatic fluid, culturedhuman prostatic malignant cells or their media are purified to obtain apreparation consisting essentially of a human prostate antigen free ofprostatic acid phosphatase. These antigenic preparations are employedfor immunological vaccination and diagnostic procedures, particularlyfor immunoprecipitin testing.

Since immunological studies indicate that this antigen is present innormal, benign hypertrophic and neoplastic prostate, a pool of variousprostate tissues can be used as the starting material for subsequentpurification. Briefly, the prostate tissue is first extracted in aqueousmedia at 4° C. Although EDTA-PBS solution is conveniently used, saline,3 M KCl or 0.01 percent (v/v) Tween 80 (but not 1 M perchloric acid)also can be used for extraction of the prostate antigen. The antigen isthus also distinguished from carcinoembryonic antigens by itssensitivity to perchloric acid.

After clarification of the homogenate by centrifugation and filtration,the supernatant from the crude tissue extract is subjected to ammoniumsulfate fractionation. Ammonium sulfate concentration at 20-80 percentsaturation almost quantitatively recovers the prostatic antigen fromEDTA-PBS extracts, with the highest yield at 45-50 percent saturation.In order to avoid contamination by other proteins as much as possible,the precipitate is preferably collected between 35-55 percent saturationof ammonium sulfate, which contains approximately 70 percent of thetotal prostate antigen in the crude extract.

Human seminal plasma has been found to contain an antigen, reacting withrabbit anti-PA serum in double immunodiffusion, which forms a fusedimmunoprecipitin line with that of crude extract of prostatic tissue.This result revealed the presence of a protein, immunologicallyidentical to the human prostate antigen (PA), in seminal plasma. Furtherstudy showed that all seminal plasma specimens examined contained asubstantial amount of PA, ranged from 0.4 to 1.8 mg/ml, (n=9,mean±standard deviation=0.71±0.42). It is estimated that PA content in20 to 30 ml of seminal plasma was equivalent to that in 100 g ofprostatic tissue. The elution profile of PA in chromatographies issimilar to that using prostatic tissue as source of the antigen. PApurified from seminal plasma and from prostatic tissue possesses anidentical mobility and isoelectric point (pI 6.87±0.09) as shown bypolyacrylamide gel electrophoresis and isoelectric focusing,respectively. Both purified PA preparations exhibit a molecular weightof 33-34,000 as shown by Sephadex G-75 gel filtration. In addition, aline of identity was obtained in immunodiffusion when purified PApreparations reacted with anti-PA serum.

Since seminal plasma is more readily available than prostatic tissuesand contains abundance of PA, it appears an ideal source for isolatingPA. Using seminal plasma as the source of PA isolation also appears tohave an advantage over the use of prostatic tissue. Firstly, the initialextraction step, which requires at least 4 hours, is eliminated.Secondly, at the initial stage of purification, handling a large volumeof the tissue extract is avoided since 20 to 30 ml of seminal plasma isequivalent to 100 g of tissue in terms of PA content. Thirdly, seminalplasma contains less contaminating proteins and makes purificationeasier. For instance, hemoglobin in the tissue extract is precipitatedconcurrently with PA by ammonium sulfate at concentration greater than50 percent saturation, and the removal of this hemoglobin in later stepsof purification results in a reduction of the yield of final purifiedPA. Since seminal plasma contains less contaminating proteins, it ispossible during fractional precipitation steps to recover a greateramount of PA by increasing the upper cut-off point of ammonium sulfateconcentration to 75 percent saturation. A better recovery of PA inpurified form is also achieved from seminal plasma.

DEAE-BioGel A anion exchange column effectively retains the prostateantigen and washing the column with tris-HCl buffer at pH 8.0 does notdissociate the antigen from the column. Elution of the antigen can beachieved with 18-78 mM of NaCl in the same buffer, followed by furtherpurification by gel chromatography. The bulk of contaminating proteinsin the PA preparation eluted from the DEAE column have a molecularweight greater than 45,000 and can accordingly be separated from PA(molecular weight: 33,000 to 34,000) by gel filtration on a SephadexG-100 column.

An additional step can be carried out with anion exchange chromatographyon a DEAE column using a pH gradient solution as the eluant. Two proteinpeaks were detected between fractions in the pH range of 7.6 to 6.7,which were shown to contain PA. Upon polyacrylamide gel electrophoresis,protein heterogeneity was seen in the first peak fractions, while thesecond peak fractions gave a single protein band. Therefore, forobtaining homogeneous PA, only second peak fractions were collected. Atthe end of theis elution, approximately 20 percent of total PA subjectedto chromatography was still bound to the DEAE column. This can berecovered quantitatively by elution with 0.08 M NaCl, but proteinheterogeneity in the eluted PA preparation was observed. An interestingobservation has been made in the course of the above PA purification;the first peak fractions contained PA which possessed a pI differentfrom that of the purified PA from the second peak fractions. Also, thePA retained by the DEAE column at the end of pH gradient elutionexhibited a different pI. Similar observations have also been made inthe purification of PA from seminal plasma, and treatment withneuraminidase has increased the pI of thse PA "isomers" to a higher pHrange. It thus appears that the human prostate antigen of the presentinvention may exist in a number of different isomeric forms.

Preparative polyacrylamide gel electrophoresis of the partially purifiedantigen is effective in achieving a pure antigen preparation. Thispurified antigen is shown to be homogeneous by polyacrylamide gelelectrophoresis with and without sodium dodecyl sulfate. It has amolecular weight of 33,000-34,000 with no subunit, and exhibits a singlepI of 6.9.

Using immunoprecipitation and immunocytochemical techniques, PA has beenshown to be a prostate gland epithelial marker protein. PA is localizedin the epithelial lining of prostatic glands and ducts as well as inprostatic secretions and concretions, but not in epithelia ofperiurethral glands, seminal vesicles, vas deferens, urinary bladdertransitional epithelium, prostatic urethra, glandular lining ofvonBrunn's nests, or in testes. These observations suggest that the PAin the seminal plasma is of prostatic origin.

Data are now available to indicate the potential clinical application ofPA in prostatic cancer. Quantitation of circulating PA, with asensitivity of 0.1 ng/ml, has been achieved by an enzyme-immunoassay. PAis not detected in sera from normal females or female cancer patients,while sera from male patients with non-prostate cancer contain a similarrange of PA as that of normal males. Patients with prostatic diseaseshave been shown to have elevated levels of circulating PA. Although noquantitative difference in PA levels is found between patients withbenign prostatic hypertrophy and stage A of prostatic cancer, patientswith other stages of prostatic cancer demonstrate significantly elevatedPA levels, both quantitatively and qualitatively. With immunocyto-chemical procedures, all primary and secondary prostatic tumors examinedreacted positively with anti-PA serum, whereas the tumors ofnon-prostatic origin did not react. These results, therefore, suggest anadditional means for diagnosis of prostate cancer and for monitoring theefficacy of its treatment. Another possible application is the use ofspecific anti-PA antibodies in in vivo radioimmunodetection of prostatecancer, e.g. according to the method described by D. Pressman in CancerResearch 40: 2960 (1980), particularly the micro-metastasis which is socritical in staging and treatment. Immune-specific chemotherapy also isa potential area where much work can now be initiated with theavailability of PA immunologic reagents, e.g. see T. Ghose et al. in J.Nat. Cancer Inst. 61: 657 (1978). Furthermore, the physiology of theprostate can be studied with the aid of this new marker for prostategland epithelium.

In order to produce antisera which can be used to detect antigens inprostate tissues and fluids other than acid phosphatase, female rabbitshave been injected with a purified prostate antigen isolated fromprostate tissue. Sera is collected, heat inactivated and stored at -20°C. until use. After treatment with insolubilized normal human plasmaproteins (antibodies to normal plasma constituents are removed bytreatment of the antiserum with glutaraldehyde-insolubilized normalplasma obtained from normal male and female adults), the antiserumreacts specifically with prostate tissue extracts (43/43) using thetechniques of double immunodifussion and rocket immunoelectro-phoresis.No immunological reactivity was observed against a battery of extractsprepared from tissues other than prostate. The prostate antigen fromprostate tissue extracts was characterized by gel filtrationchromatography (m.w. 30-40,000), isoelectric focusing (pI 6.9) andagarose electrophoresis (M_(R) 0.2 relative to bovine serum albumin).The concentration of the prostate antigen was not significantlydifferent among extracts prepared from normal, benign hypertrophic andmalignant prostatic tissues and the antigen exhibited no acidphosphatase enzyme activity as determined by histochemical stainingprocedures. Furthermore, its approximate molecular weight (30-40,000)differs significantly from that of prostatic acid phosphatase (100,000).

Although the antigen detected in serum had a higher apparent molecularweight (80-100,000), antigen mixing and peak enhancement experimentsindicate that prostate antigen in circulation is immunologicallyidentical to the human prostate antigen in prostate tissue and prostaticfluid. The serum-borne antigen may be bound to a plasma protein. Suchplasma protein binding of a variety of antigens and hormonal substancesis well known and has been reported by T. Peters, Jr. "Serum Albumin"In: F. W. Putman (ed.). The Plasma Proteins. Vol. 1: pp. 133-153, NewYork, N.Y. Academic Press, 1975. However, SPD-PAGE revealed a molecularweight of 36,000, similar to that in prostate tissue and seminal plasma.

Alternatively to the conventional techniques for preparing antibodies inlaboratory and farm animals, monoclonal antibodies against PA can beprepared using known hybridoma cell culture techniques. In general, thismethod involves preparing an antibody-producing fused cell line, e.g. ofprimary spleen cells fused with a compatible continuous line of myelomacells, and growing the fused cells either in mass culture or in ananimal species from which the myeloma cell line used was derived or iscompatible. Such antibodies offer many advantages in comparison to thoseproduced by innoculation into animals, as they are highly specific andsensitive and relatively "pure" immunochemically.

The full potential of this prostatic antigen in clinical applications isnot known at present, as it does not appear to exhibit any tumorspecificity or pathological association. However, as in the case of acidphosphatase which is present in all prostate tissues, this prostateantigen can provide useful clinical information upon serum monitoring.Recent experiments in our laboratory have shown that the prostateantigen is detected, by rocket immunoelectrophoresis and enzyme-linkedimmunoassay techniques, in the sera of some prostate cancer patients butnot in the sera of normal healthy persons or of patients with othercancers. Further experiments have shown that PA is histologicallydetectible in both prostate tissue and established continuous tissueculture cell lines of prostate origin, and that it is released byprostate tumor cells, both in vivo and in vitro. In addition, it hasbeen found that the PA level in serum of patients suffering fromprostatic cancer is unrelated to the serum levels of prostatic acidphosphatase, so that simultaneous determination of circulating PA andcirculating prostatic acid phosphatase may well provide an improvedmeans of diagnosing prostatic cancer. The human prostate antigen,although an eutopic component of the prostate, can thus play a majorrole in the detection of prostate cancer.

For the preparation of immunogens suitable for preparing diagnosticantibodies against the human prostate antigen, conventional vaccinepreparation techniques can be used. Preferably a non-antigenic adjuvant,e.g. alum, Freund's complete adjuvant, saponin, a quaternary ammoniumsurfactant, an alkyl amine, etc. is admixed with the purified prostateantigen in a suitable immunologically acceptable, non-antigenic carrierand the resultant mixture can be sterilized, e.g. by filtration.

The vaccine can be administered parenterally following regimens alreadyknown for immunization with other proteins to stimulate the formation ofimmunoprecipitating antibodies, with the primary inoculation beingpreferably followed up by at least one additional injection one to tenweeks later. Good results have been obtained in rabbits using fourbooster injections at two week intervals one month after the primaryimmunization. The protein content per injection in rabbits, goats andother mammals can be varied, but is generally about 50 micrograms ofprotein per kg. of body weight. The antibodies can be collected andworked up using methods well known to those skilled in the art ofimmunochemistry, and provide a useful reagent for the immunologicaldetection of prostate specific antigen in a variety of immunochemicalprocedures, e.g., immunoprecipitin, fluorescent antibody, serumneutralization, etc. Such antibodies are useful as a control reagent inthe diagnostic test for prostatic cancer described more particularlybelow.

The simplest immunoprecipitin test involves capillary tube precipitintesting, wherein separate antibody and antigen solutions are allowed toreact at a common interface in a capillary tube and a positive reactionis indicated by the formation of a precipitate at the interface. Thismethod is relatively insensitive and inaccurate due, inter alia, tounavoidable diffusion of the two solutions across the interface, andfurthermore the final test results cannot be preserved.

Agar gel diffusion is the simplest method which avoids these drawbacks.A solution of the antigen (or serum sample) is placed in a central wellpunched in a continuous agar gel and appropriate dilutions of the serumcontaining antibodies (or, correspondingly, the antigen) thereto areplaced in wells concentrically surrounding the center well. A positivereaction is noted by the formation of the precipitin line between one ormore of the concentric wells and the central well. This method isrelatively insensitive and fairly slow, requiring 1 to 4 days to readthe test results.

Radioimmunoassay (RIA), e.g. radioimmunoprecipitin tests, are extremelysensitive (by several orders of magnitude over older methods) but takeseveral days to perform and require sophisticated equipment and highlytrained personnel not always widely available.

Countercurrent immunoelectrophoresis (CIEP) is a widely usedimmunoprecipitin method which takes only about an hour to perform andwhich is considerably more sensitive than agar gel diffusion. Reactivecomponents are placed in opposing wells cut into an agar gel and a smallelectrical current applied thereto, causing both the antigen and theantibody to migrate towards each other. A positive reaction is indicatedby the formation of a precipitate at the antigen-antibody interface.Since this test method is reasonably reliable, readily available andinexpensive, it represents a preferred embodiment of this aspect of thepresent invention.

For purposes of immunoelectrophoresis testing, the diagnostic antibodypreparation of the present invention when used without purification isgenerally diluted with phosphate buffered saline in a volume ratio of1:10 to 1:500, depending on the antibody titer thereof. The limitingfactor at the lower end of the range is the degree of distinctionachieved in the precipitin lines, which is a function of the antibodycontent in the total protein present. Purified antibody preparations canof course have lower total protein concentrations, and the proteincontent of even the unpurified preparations can be varied to suit theparticular immunochemical test to be employed, the optimal amounts beingdetermined, e.g. by testing simple serial dilutions.

In a preferred, further aspect of the present invention, circulatinghuman prostate antigen can now be detected by immunochemical techniques,preferably by protein staining of the antibody-antigen precipitincomplex. Using the aforementioned specific antisera and coupling thiswith a conventional means for detecting the antigen-antibody complex, itis now possible to immunochemically separate the specific human prostateantigen from a serum sample.

The antigen-antibody complex can be stained by a number of knownhistochemical staining techniques, e.g. fluorescent antibody, etc., toincrease the sensitivity of this method. Alternatively, one can useradioactive antibody for the assay, which not only provides a betterquantitative value but may also further increase the sensitivity of theassay. If desired, an enzyme, e.g. β-galactosidase or peroxidase, can becoupled with purified antibodies for use in an enzyme-linkedimmunoassay, e.g. using techniques analogous to those described by Katoet al. in J. Immunol. 116: 1554 (1976), the contents of which areincorporated by reference herein. Especially preferred is the methoddescribed by M. Kuriyama et al. in Cancer Res. 40: 4658 (1980), thecontents of which are incorporated by reference herein.

It is preferable to bind these antibodies onto a water-insoluble supportfor use in the enzyme assay. Many suitable such supports and techniquesfor binding proteins thereto are well known in the art and includeinorganic as well as organic supports. Presently preferred are thosewater-insoluble supports which can be activated with a cyanogen halide,preferably cyanogen bromide, prior to the covalent bonding of theantibodies thereto, e.g. as taught by Axen et al. in U.S. Pat. No.3,645,852, the contents of which are incorporated by reference herein.Such supports are commercially available, e.g. the Enzymobeads availablefrom Bio-Rad Laboratories.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following preferred specific embodiments are,therefore, to be construed as merely illustrative and not limitative ofthe remainder of the disclosure in any way whatsoever. In the followingExamples, the temperatures are set forth uncorrected in degrees Celsius;unless otherwise indicated, all parts and percentages are by weight.

Protein concentration was determined by the method of Lowry et al.described in J. Biol. Chem. 193: 265 (1951), using bovine serum albuminas the standard. Acid phosphatase activity was measured by the method ofBabson and Phillips using α-naphthyl phosphate as the substrate.

Immunoprecipitation techniques were modified from B. Weeke, Scand. J.Immunol. (Supplement 1) 2: 37-46 (1973). Rocket-IEP was performed oncellulose acetate membranes using 0.83 percent agarose (Sigma lowelectro-endo-osmosis) in 0.08 M tris-0.024 M tricine-0.024M sodiumbarbital containing 1.3 mM calcium lactate and 0.02 percent sodium azide(TTB buffer). Antiserum at various final concentrations (0.5-2.0percent) was incorporated into the agarose at 55° C. prior to plating.Rocket-IEP samples were applied to circular wells (5.0 mm) andelectrophoresed at 5 volts/cm overnight at 4° C. using TTB runningbuffer.

Crossed-immunoelectrofocusing was performed in the second dimension asfor rocket-IEP. Isofocused acrylamide gels (5×100 mm) were molded intothe antibody-agarose prior to immunoelectrophoresis modified from J.Soderholm et al., Scand. J. Immunol. (Supplement 2) 4: 107-113 (1975).The first dimension isofocusing was done in 7.5 percent polyacrylamidecontaining 2 percent ampholytes, pH range 4-8 at 200 volts overnight at4° C. For some experiments, isofocused gels were laid on celluloseacetate membrances and covered with 1 percent agrose. Troughs were cutadjacent to the embedded acrylamide gel, the antiserum placed in theparallel troughs was allowed to diffuse for 24-48 hours at 25° C. andthe resulting immunoprecipitin lines were recorded.

Crossed-immunoelectrophoresis (cross-IEP) was performed in the seconddimension as for rocket-IEP. The first dimension electrophoresis wasperformed with 1 percent agarose, and samples (75 μl in rectangularwells) were electrophoresed at 15 volts/cm for 2 hours at 4° C.Migration was monitored with Bromphenol Blue dye.

The abbreviations used are: PB-NaCl, 0.05 M phosphate buffer containing0.15 M NaCl, pH 7.2; IEP, immunoelectrophoresis; TTB,Tris-Tricine-Barbital (0.08M, 0.042M, 0.024M) buffer containing 0.3 mMcalcium lactate and 0.02 percent sodium azide, pH 8.8; M_(R), relativeimmunoelectrophoretic mobility; pI, isoelectric point; m.w., molecularweight.

EXAMPLE 1 Extraction of Tissues

Human prostatic tissues (normal, benign hypertrophic, malignant) andother human tissues were obtained during autopsy or surgery. Thehistology of each issue was confirmed by pathologists. Approximately 10g of tissues were minced and washed three times with 30 ml ofphysiological saline, then mixed with 30 ml of 0.02 percent (w/v)disodium ethylenediamine tetraacetate (EDTA)-0.1M phosphate bufferedsaline (PBS), pH 6.8. The mixture was placed in an ice-water-chilledblending chamber of a Sorvall Omnimixer and subjected to three 5-minuteblendings at a blade speed of 25,000 rpm with intermittent cooling timeof 3 minutes. The homogenate was stirred overnight at 4° C., thencentrifuged at 25,000 g for 30 minutes. The resultant supernatantconstituted the crude tissue extract.

EXAMPLE 2 Ammonium Sulfate Fractionation

Pooled prostate tissues, 100 g, were extracted with 300 ml of EDTA-PBSas described above. Ammonium sulfate (60 g) was added to 285 ml of crudeextract (35 percent saturation), mixed for 30 minutes and centrifuged(26,000 g, 30 minutes). 38 g of ammonium sulfate were added to 295 ml ofthe resulting supernatant, (55 percent saturation), mixed for 30 minutesand centrifuged. The precipitate was dispersed in 200 ml of 55 percentsaturated ammonium sulfate solution and centrifuged. Washing of theprecipitate was repeated twice, then the precipitate was dissolved in 20ml of 0.01M tris-HCl buffer, pH 8.0. The residual ammonium sulfate wasremoved by dialysis against 4 liters of the tris-HCl buffer for 48 hourswith one change of buffer during dialysis. The dialyzed solution wascentrifuged at 46,000 g for 30 minutes to remove any precipitate formedduring dialysis.

EXAMPLE 3 Ion Exchange Chromography of Prostate Antigen

The supernatant from Example 2, 28 ml, was applied onto a DEAE-BioGel Acolumn (2.5×93 cm) pre-equilibrated with 0.01M tris-HCl buffer, pH 8.0.The column was first washed with 420 ml of buffer, then eluted with0--0.2M NaCl gradient (1 liter 0.01M tris-HCl buffer, pH 8.0 in mixerand 1 liter of 0.2M NaCl 0.01M tris-HCl, pH 8.0 in the reservoir) at aflow rate of 6.5 ml/hr/cm². The effluent was collected (10 ml/fraction)and monitored for the prostate antigen. Gel diffusion according to themethod of T. M. Chu et al. in Cancer Treat. Reports 61: 193 (1977) wasused to measure the presence of prostate antigen in variouschromatographic fractions. The entire procedure was carried out at 4° C.The prostate antigen-containing fractions were pooled and concentratedwith an Amicon concentrator fitted with a UM2 ultrafiltration membraneto 4.5 ml.

EXAMPLE 4 Gel Filtration of Prostate Antigen

A portion (4.3 ml) of the concentrated solution was applied onto aSephadex G-100 column (2.5×110 cm) pre-equilibrated with 0.01M tris-HClbuffer, pH 8.0, which was eluted with same buffer at a flow rate of 3.5ml/hr/cm². The pooled DEAE-BioGel A fractions 100-150, containing theprostate antigen upon Sephadex G-100 chromatography, were resolved intoseveral protein peaks. The prostate antigen was found to be in fractions46-56. Again, the fractions exhibiting prostate antigen reactivity werepooled and concentrated to 3.8 ml.

EXAMPLE 5 Further Purification of Prostate Antigen

The above fractions containing prostate antigen were applied onto aSephadex G-75 column (2.5×113 cm) pre-equilibrated with tris-HCl buffer;the column was eluted at a flow rate of 5.5 ml/hr/cm². This resulted inone major symmetrical protein peak associated with the prostate antigenreactivity and two minor protein peaks. When the fractions containingthe prostate antigen were re-chromatographed on the same column, anidentical symmetrical protein peak was obtained and the minor proteinpeaks were eliminated. Acid phosphatase activity was measuredcolorimetrically and histochemically as described by Babson et al. inClin. Chim. Acta 13: 264-265 (1966) using α-naphthyl phosphate as thesubstrate. No prostatic acid phosphatase was detected in thechromatographic fractions of this protein peak.

EXAMPLE 6 Analytical Polyacrylamide Electrophoresis

Acrylamide gel (7.5 percent) columns (50×60 mm) were made according tothe company's instruction manual (Shandon Southern Instruments, Ltd.,Camberley, England), and 50 μl of sample (10-40 μg protein in 25 percentsucrose solution) were applied onto each gel column. Ten percent sucrosesolution was then layered carefully above the sample solution, followedby layering 0.05 M tris-glycine buffer, pH 8.5 to the top of thegel-containing tube. Tris-glycine buffer was used in preparing thesucrose solutions and used also as the electrolyte. Afterelectrophoresis with a constant current of 5 mA per tube for 40 minutes,the gels were stained for protein by Coomassie brilliant blueG-250-perchloric acid solution.

EXAMPLE 7 Sodium Dodecyl Sulfate (SDS) Polyacrylamide GelElectrophoresis

The method was essentially the procedure or Weber and Osborn describedin J. Biol. Chem. 244: 4406 (1969). A sample (10-20 μg protein) in 50 μlof 0.05M tris-glycine buffer, pH 8.5, containing 250 μl SDS and2-mercaptoethanol each, was incubated at 37° C. for 2 hours. Afterincubation, the sample was mixed with an equal volume of 50 percentsucrose, and 50 μl were subjected to the polyacrylamide gel (containing0.1 percent SDS) electrophoresis as described before. Afterelectrophoresis, the gels were stained for protein with 0.5 percent(w/v) Coomassie brilliant blue G-250 in ethanol-acetic acid-water(45:10:45, v/v).

EXAMPLE 8 Preparative Polyacrylamide Gel Electrophoresis

Although Sephadex G-75 chromatography produced a symmetrical proteinpeak with exhibited immunological reactivity of the prostate antigen,the analytical polyacrylamide gel electrophoresis of Examples 6 and 7revealed several protein components in this preparation. Therefore, apreparative polyacrylamide gel electrophoresis (PAGE) was further usedin the purification of prostate antigen.

One ml of specimen (4.5 mg protein) was mixed with 1 ml of 50 percent(w/v) sucrose solution and applied onto an annular 7.5 percent gelcolumn (cross-sectional gel area, 4.4. cm² ; height, 9.2 cm), followedby successive layerings of 2 ml of 10 percent sucrose and 0.05Mtris-glycine buffer, pH 8.5. A constant current of 30 mA was firstapplied for 1 hour, and 80 mA thereafter. The tris-glycine buffer wascontinuously pumped at a flow rate of 14-15 ml/hour into the elutionplate located at the bottom of the column to carry materials emergedfrom the column into a fractional collector. Fractions containing theprostate antigen, representing the final purified preparation, werepooled and concentrated to 0.5 ml. The prostate antigen was found infractions 28-62 (eluates obtained between 4-9 hours afterelectrophoresis). When these fractions were pooled and subjected toanalytical polyacrylamide gel electrophoresis, multiple protein bandsstill existed. However, by pooling fractions 56-62 (between 8.1-9.2hours of electrophoresis) only a single protein band was obtained uponanalytical polyacrylamide gel electrophoresis and sodium dodecyl sulfatepolyacrylamide gel electrophoresis.

That the final prostate antigen preparation was of high purity wasindicated by a single protein band without any contaminating componentsin polyacrylamide gel electrophoresis in the absence and the presence ofsodium dodecyl sulfate. The purified prostate antigen was shown to havea molecular weight of 33,000 by gel filtration on Sephadex G-75, and34,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis withno subunit component. Isoelectric focusing revealed a single pI of 6.9.

EXAMPLE 9 Simplified Purification of Antigen from Tissue

The procedure for the purification of PA reported in the precedingExamples takes at least two weeks and yields less than 0.5 mg ofpurified PA per 100 g of prostatic tissue. Furthermore, for the finalstep of purification, a painstaking construction of the polyacrylamidegel column as well as a special electrophoretic apparatus are necessary,and problems such as clogging of the elution channel during operationoften developed. In this simplified procedure, in addition to theelimination of Sephadex G-75 chromatography and the application of shortDEAE columns, the tedious preparative gel electrophoresis has beenreplaced by a simple pH gradient elution of the DEAE column. The yieldof purified PA has increased to 1 to 2 mg per 100 g of prostatic tissueand the entire procedure has been shortened to 8 to 9 days.

Benign hypertrophic prostatic tissues (approximately 100 g) wereextracted with disodium tetraacetate-phosphate buffered slaine asdescribed in Example 1 except that the homogenate was stirred for only 2hours rather than overnight. The crude extract was then subjected tofractional precipitation by ammonium sulfate (30 to 50 percentsaturation). The precipitate was dissolved in 10 to 15 ml of 0.01Mtris-HCl buffer, pH 7.8, and dialyzed against 4 ml of the same bufferfor 48 hours with one change of buffer. The dialyzed materials werecentrifuged (46,000 g, 30 min) and the supernatant (12 to 18 ml) wasapplied onto a 2.5×15 cm DEAE-BioGel A column pre-equilibrated with0.01M tris-HCl buffer, pH 7.8. The column was washed with ca. 300 ml ofthe same buffer, followed by elution with 0.08 M NaCl-0.01M tris-HClbuffer, pH 7.8. Fractions containing PA were pooled, concentrated (5 to5.5 ml), and applied to a 2.5×100 cm Sephadex G-100 columnpre-equilibrated with 0.01M tris-HCl buffer, pH 7.8, which was theneluted with the same buffer at a flow rate of 5 ml per hour per cm².Fractions covering a molecular weight region of 26,000 to 37,000 whichcontained PA were pooled, concentrated (5 ml) and applied to a 2.5×20 cmDEAE column pre-equilibrated with 0.01M tris-HCl buffer, pH 7.8. Thecolumn was washed with 10 ml of the same buffer, followed by elutionwith pH gradient solution (mixer: 0.01M tris-HCl, pH 7.0, 300 ml;reservoir: 0.01M tris-HCl, pH 6.0, 500 ml) at a flow rate of 5 ml perhour per cm². Second peak fractions which contained PA were pooled andconcentrated.

Table 1 summarizes the results of a typical purification of PA fromprostatic tissue by this simplified procedure. Although modification atthe salt precipitation step led to lesser recovery of PA, a largequantity of contaminating proteins such as hemoglobin were removed,thereby facilitating further purification of PA. Radial immunodiffusionused in this study was the technique of Mancini et al described inImmunochem. 2: 235-254 (1965), with modifications. Anti-PA serum (100μl) was mixed with 10 ml of 1 percent agarose solution (in 0.154MNaCl-0.017M solium phosphate, pH 7.0) at 55° C. and poured into a Petridish (diameter: 8.3 cm). Wells (diameter: 2 mm) were then made on theagarose gel and to each well, 10 μl of sample was added. For monitoringPA in the chromatographic fractions, overnight diffusion was sufficientto reveal precipitin; for quantitation of PA, the diffusion was allowedto continue for 48 hours, followed by washing of the gel for 2 days with0.154M saline and subsequent staining with Coomassie Brilliant BlueG-250.

                  TABLE 1                                                         ______________________________________                                        Purification of Prostate Antigen from                                         Human Prostatic Tissue                                                                                                 Recov-                                           Total   Total    Total Folds ery                                              volume  protein  PA    purifi-                                                                             (per-                                Steps       (ml)    (mg)     (mg)  cation                                                                              cent)                                ______________________________________                                        Crude extract.sup.a                                                                       258.0   1754.4   20.6  1     100                                  Ammonium sulfate                                                                          13.8    278.8    10.0  3.1   48.5                                 (30-50 percent)                                                               First DEAE  5.5     80.9     7.9   8.3   38.3                                 Sephadex G-100                                                                            5.0     9.3      4.3   39.4  20.9                                 Second DEAE 5.0     1.5      1.5   85.2  7.3                                  ______________________________________                                         .sup.a From 118 g of benign hypertrophic prostate tissues.               

EXAMPLE 10 Purification of Antigen from Fluid

PA was also purified from 20 ml of seminal plasma according to theprocedure of Example 9 except that the extraction step was omitted andthat, in the second step, PA was precipitated by ammonium sulfate at aconcentration of 30 to 75 percent saturation. The results are shown inTable 2.

                  TABLE 2                                                         ______________________________________                                        Purification of Prostate Antigen from                                         Human Seminal Plasma                                                                      Total   Total   Total Folds Recov-                                            volume  protein PA    purific-                                                                            ery                                   Steps       (ml)    (mg)    (mg)  cation                                                                              (percent)                             ______________________________________                                        Seminal plasma                                                                            16.2    595.2   8.8   1     100                                   Ammonium sulfate                                                                          20.0    266.2   7.4   1.9   84.1                                  (30-75 percent)                                                               First DEAE  5.3     61.0    5.7   6.3   64.8                                  Sephadex G-100                                                                            6.4     12.8    4.5   23.8  51.1                                  Second DEAE 4.5     1.5     1.5   67.6  17.0                                  ______________________________________                                    

EXAMPLE 11 Molecular Weight Determination

In order to obtain an approximate molecular weight of the prostateantigen present in crude prostate tissue extracts and inantigen-positive patients' sera, these samples were subjected to aSephadex G-200 gel filtration chromatography. Serum samples (0.5 ml)previously shown to be prostate antigen-positive by rocket-IEP, orprostate tissue extracts (0.5 ml containing 8 mg protein) were appliedto a column (0.9×60 cm) packed with Sephadex G-200 gel in PB-NaCl.Eluted samples were analyzed for absorbance at 280 nm and prostateantigen level was determined by rocket-IEP using a sample size: 0.5 ml;equilibration buffer: PB-NaCl (pH 7.2); fraction size; 0.8 ml; elutionrate: 10 ml per hour. Peak antigen activity as exhibited in prostatetissue extracts eluted between 30-40,000 m.w. Molecular weight referencemarkers included human immunoglobulin G (160,000), bovine serum albumin(68,000), ovalbumin (43,000), chymotrpysinogen A (25,000) andribonuclease A (13,700). In antigen-positive patient sera examined bygel filtration, prostate antigen eluted as a single symmetrical peakbetween 80- 100,000 m.w.

EXAMPLE 12 Preparation of Antisera

Female rabbits were immunized as described previously by T. M. Chu etal. in Investigative Urology 15: 319-323 (1978) with the crude extractof normal human prostatic tissue (for antiserum P₈), or with a purifiedprostate antigen (for antiserum P₁₇) obtained at the Sephadex G-75 stepdescribed above. Sera were collected, heat inactivated and stored at-20° C. until use. Absorption of the antiserum with normal female serum(NFS) or tissue extracts (10 mg protein/ml) was carried out as describedby T. M. Chu et al. in Cancer Treatment Reports 61: 193-200 (1977).

EXAMPLE 13 Specificity of Antiserum Raised Against Crude ProstateAntigen

Immunoelectrophoresis was performed on a 9.5×10.2 cm agarose (0.65percent, w/v) plate. Barbital buffer, (pH 8.2, ionic strength 0.04) wasused as the electrolyte and a constant voltage of 90 V. was applied forone hour. After electrophoresis and gel diffusion (20 hours), the platewas washed with 0.154 M saline for 2 days and stained first for acidphosphatase with a solution of α-naphthyl phosphate-fast garnet GBC saltin 0.1M ammonium acetate, pH 5.0, and then for protein with Coomassiebrilliant blue G-250-perchloric acid solution.

Immunoelectrophoresis of the crude extract prepared from normalprostatic tissues and antiserum P₈, an antiserum raised against thecrude extract of normal prostate, resulted in three precipitin arcs. Oneof these arcs was formed by a normal human serum component, as itdisappeared after the antiserum was absorbed with normal female serum(NFS-P₈). Absorption of the antiserum NFS-P₈ with various normal humantissue extracts (urethra, bladder, heart, lung, pancrease, bone, kidney,intestine, liver and spleen, 10-20 mg each per ml) failed to eliminatethe two remaining precipitin arcs, one of which was identified asprostatic acid phosphatase since it was stained with the α-naphthylphosphate-fast garnet GBC salt solution. The other precipitin arc wasshown to be a prostate tissue-specific antigen, identified as a protein,not stainable for acid phosphatase activity, which migrated withβ-mobility upon IEP analysis. Absorption with an extract of normal humanprostate removed the reactive antibodies from the antiserum NFS-P₈ andabolished both precipitin arcs. Furthermore, this antigen wasdemonstrated in all 20 of 20 normal prostates and identical results wereobtained with the extracts of benign hypertrophic (15/15) and cancerousprostatic (8/8) tissues. These data demonstrate that normal, benignhypertrophic and malignant prostate contain a prostatic tissue-specificantigen in addition to prostatic acid phosphatase.

EXAMPLE 14 Specificity of Antiserum Raised Against Purified ProstateAntigen

Following the procedure of Example 11, additional confirmation wasprovided with the use of antiserum P₁₇ raised against a purifiedpreparation of the prostate antigen, which was devoid of prostatic acidphosphatase. Both gel diffusion and immunoelectrophoresis employing thenormal adult male and female serum-absorbed antiserum P₁₇ resulted in asingle immunoprecipitation of the prostate antigen. The extractsprepared from tissues other than the prostate did not react with thenormal female serum-absorbed antiserum P₁₇.

EXAMPLE 15 Immunological Identity of Tissue and Serum Prostate Antigen

In order to determine if the serum-borne antigen shared immunological orbiochemical characteristics with the antigen detected in prostate tissueextracts, the following experiments were performed. Samples of sera andprostate tissue extracts were subjected to rocket-IEP both individuallyand immediately after combination of both antigen sources. In eachexperiment, the samples were adjusted to the same final proteinconcentration by the use of buffer dilutions. A representativeexperiment employed two sample wells containing patient sera, samplewells containing prostate tissue extract and two sample wells containinga mixture of patient's serum and tissue extract to demonstrate peakenhancement. Sera and tissue extracts were at the same final proteinconcentration in each well by the use of appropriate volumes of dilutingbuffer (PB-NaCl). All samples were 25 μl; one percent antibody in 0.83percent agarose; 5 V per cm, 20 hours at 4° C. All samples produced asingle immuno- precipitate when assayed individually. Mixing samples, inpeak enhancement experiments, produced a single reaction whose heightwas greater than that of individual samples. When antigen-positive serumsamples and prostate tissue extracts were mixed and immediatelysubjected to crossed-IEP, a fused immunoprecipitation peak resulted.Both methods of quantitative immunoelectrophoresis, showing peakenhancement and immunoprecipitate fusion, confirmed the immunologicalidentity of the prostate antigen as it occurs in tissue and serumaccording to the method of N. H. Axelson et al. in Scand. J. Immunol.(Supplement 1) 2: 91-94 (1973).

It should be noted that results regarding prostate antigen, as detectedin tissue, apply to extracts prepared from normal, benign hypertrophicand malignant prostate specimens; no physical differences in prostateantigen (i.e., M_(R), pI or m.w.) were observed among these antigensources, and extracts prepared from benign and malignant prostatespecimens exhibited immuno-precipitation lines of identity with normalprostate when tested against anti-prostate antigen antiserum by doubleimmunodiffusion. In addition, the relative level of prostate antigen wascompared among extracts prepared from normal, benign hypertrophic andmalignant prostate tissues. As determined by rocket-IEP (Table 3) nostatistically significant difference of antigen level was observedbetween these antigen sources and a wide variation in antigen level(indicated by the large standard deviations calculated) was also notedto occur for each category of tissue extract examined.

                  TABLE 3                                                         ______________________________________                                        PROSTATE ANTIGEN LEVELS IN PROSTATE                                           TISSUE EXTRACTS.sup.a                                                         Tissue Pathology.sup.b (Number)                                                                Prostate Antigen Level ± S.D.                             ______________________________________                                        Normal (4)       14.3 units.sup.c (± 8.7)                                  Benign hypertrophic (8)                                                                        18.4 units.sup.c (± 21.3)                                 Primary carcinoma (8)                                                                          13.0 units.sup.c (± 13.8).sup.d                           ______________________________________                                         .sup.a Each tissue extract was adjusted to 1 mg protein per ml prior to       analysis for prostate antigen by rocket immunoelectrophoresis.                .sup.b All tissue specimens were histopathologically confirmed.               .sup.c For this experiment, one unit was arbitrarily chosen to represent      one percent of the antigen level of a reference prostate extract. The         reference extract was asssyed in a dilution series and run on the same        plates as experimental extracts.                                              .sup.d As analyzed using Student's ttest, no statistically significant        difference was found among these groups.                                 

EXAMPLE 17 Immunological Identity of Fluid and Serum Prostate Antigen

Since prostatic acid phosphatase has been reported in both prostatictissue and seminal fluid, we wished to determine whether PA also existsin the seminal fluid. Anti-PA serum, raised against PA isolated fromprostatic tissue, reacted in immunodiffusion with crude extract ofprostatic tissue, as well as with seminal plasma, and formed a fusedline, indicating immunologic identify of the PA in prostatic tissue withthat in seminal plasma. By the previously described radialimmunodiffusion procedure, the PA concentration in seminal plasma wasfound to be 0.4 to 1.8 mg/ml, while 1 g. of prostatic tissue contained0.15 to 0.45 mg of PA extractable with disodium ethylenediaminetetraacetate-phosphate buffered saline (EDTA-PBS).

EXAMPLE 18 Histological Localization of Prostate Antigen

Localization of PA in the prostate gland as well as specificity of PAhave been probed with the immunoperoxidase staining technique ofHeyderman and Neville reported in J. Clin. Path. 30: 138-143 (1977).Sections of freshly fixed prostatic tissue were deparrafinized in twochanges of xylol, rehydrated thru a graded series of ethanol, and washedwith distilled water. Following the inhibition of endogeneous peroxidaseactivity by 7.5 percent H₂ O₂, the reactivity of anti-PA was assessedwith rabbit anti-PA serum. After incubation and subsequent washing, thetissue sections were further incubated with peroxidase- labelled goatanti-rabbit γ-globulin. Excessive enzyme-labelled second antibody wasthen removed by washing, and the tissue sections were stained forperoxidase activity. As controls, the substrate alone, substrate plusconjugate, pre-immune rabbit serum, and anti-PA preabsorbed withspecific antigen were used.

It was observed that the staining was restricted to epithelial cellscomprising the prostatic ductal elements. An intense staining was shownin the apical cytoplasma of these cells, but no staining was seen in thenuclei. Within several ductal elements, positively staining secretorymaterial was observed in the section examined. Specific staining was notobserved for other cellular elements, including stromal and vascularelements. Using the same technique, PA could not be detected in sectionsderived from other organ tissues, including the pancreas, colon,stomach, liver, seminal vesicles, and testes. Employing similarimmunohistochemical techniques, PA has been demonstrated in all primaryand metastatic prostatic tumors tested, but not in non-prostatic cancertissues.

EXAMPLE 19 Detection of Prostate Antigen in Established Tissue CultureCell Lines

To determine if malignant cells in long-term culture retain theexpression of PA, three established strains of prostate cells (LNCaP,PC-3, and Du-145) were examined. Extracts prepared from these cells andother cultured cells of nonprostate origins were assayed for thepresence of PA by the previously mentioned Enzyme Immunoassay (EIA)procedure capable of detecting 1 ng PA/ml.

Immobilized anti-PA was prepared using CNBr-activated Sepharose 1B(Pharmacia Fine Chemicals). The reaction mixture consisted of 5 g ofCNBr-activated Sepharose and 130 mg of IgG (anti-PA) in 0.1M boratebuffer (pH 8.5) containing 0.5M NaCl. After incubation at 4° C. for 18hours, the beads were washed with borate buffer and post-treated with a1-M ethanolamine solution (pH 9.0) to block unreacted groups. Covalentlycoupled beads were further washed and stored in PBS at 4° C.

For the EIA, 100 μl of an antigen sample was mixed with 300 μl of afifty fold-diluted immobilized antibody and incubated for 3 hours atroom temperature. After the addition of 1 ml of assay buffer (PBScontaining 1 percent BSA), the mixture was centrifuged at 1,000 g towash the beads. This procedure was repeated twice. To the washed beadswas added 100 μl of peroxidase-IgG (anti-PA) conjugate in assay buffer.After a further incubation for 18 hours at room temperature, the beadswere again washed as described above and assayed for the amount of boundperoxidase activity present. The reaction mixture contained 0.08 percentdianisidine and 0.003 percent H₂ O₂ in 0.01 M sodium phosphate buffer(pH 6.0 ) and was allowed to react with the beads for 90 minutes. Theenzyme reaction was then stopped with 100 μl of 1 N HCl, and theabsorbance at 403 mn was determined. Each run included standards ofknown prostate antigen concentration. Using this assay procedure,linearity of of the dose-response curve was achieved between 1 and 20 ngPA/ml. Before reexamination, samples containing higher levels of antigenwere serially diluted with assay buffer. The results indicated that twolines of prostate cancer cells, LNCaP and PC-3, contained significantlevels of PA (50-700 ng/ml) as compared with those found in Du-145 andother cell lines examined (less than 4 ng/ml). PA was also present inconditioned "spent" media derived from prostate cultures producing theantigen.

EXAMPLE 20 Immunological Reactivity of Cultured Prostate Cells withImmunoglobulin Antiserum Fragments

To further assess antibody specificity, we examined cultured cells fortheir ability to specifically accrete radiolabeled antibody fragments.Included in these experiments were cells derived from the prostate gland(LNCaP), colon (HT-29), and breast (MCF-7). For the preparation ofimmunoglobulin fragments, samples of IgG, anti-PA and rabbit pre-immuneserum were dissolved in 0.1M sodium acetate buffer (pH 4.0). For every50 mg of IgG, 1 mg of crystallized pepsin was added and the reactionmixture was incubated overnight at 37° C. F(ab')₂ fragments wereseparated from unreacted IgG and from smaller peptides by gel filtrationover Sephacryl S-300 (Pharmacia Fine Chemicals) equilibrated in PBSbuffer. The column (2.6×70 cm) was calibrated by the chromatography ofmolecular weight standards, including IgG, BSA, and egg albumin.Isolated F(ab')₂ fragments were concentrated by ultrafiltration with theuse of an Amicon PM-10 membrane and positive pressure.

Trace labeling of F(ab')₂ fragments was accomplished with the use ofsolid-phase lactoperoxidase-glucose oxidase (Enzymobeads; Bio-RadLaboratories). To 1 mg of F(ab')₂ were added 50 μl of Enzymobeads, 25 μlof 1 percent β-D-glucose, 200 μl of 0.2 M sodium phosphate (pH 7.0), and1 mCi of carrier-free ¹³¹ I or ¹²⁵ I. The reaction was allowed toproceed for 30 minutes at room temperature. Unreacted iodide wasseparated from labeled protein by gel filtration over Sephadex G-25.Specific activities of trace-labeled F(ab')₂ preparations ranged between0.6 and 0.8 μCi/μg.

Cells grown in 24-well culture dishes with each well containing 1.5×10⁶cells were incubated with a paired radiolabeled mixture of ¹²⁵ I-labeledF(ab')₂ (anti-PA) and ¹³¹ I-labeled F(ab')₂ (preimmune) in fresh media.This mixture contained 1.5×10⁵ cpm for each radioactive nuclide and wasallowed to react with cultured adherent cells overnight under standardconditions. To determine radioactive uptake, cells were scraped from thewells and washed three times in PBS before counting for each radioactivenuclide in a Packard automatic γ-scintillation counter. Preferentialuptake of a specific radioactive nuclide was calculated as alocalization ratio: (¹²⁵ I cell bound ¹³¹ I cell bound): (¹²⁵ I added¹³¹ I added). A specific localization ratio was calculated for each celltype, and the results indicated a significantly higher antibody uptakeby the LNCaP cells of prostate origin than for the HT-29 or MCF-7 cells.

EXAMPLE 21 Release of Prostate Antigen by in vivo Tumor Cells

When established as tumor cell xenografts in nude mice, LNCaP cellsreleased detectable levels of PA into the circulation of these animals.Congenitally athymic nude mice homozygous for the nu/nu allele were bredat Roswell Park Memorial Institute from matings of BABL/c nu/nuhomozygous males and BALB/c +/nu heterozygous females. Human tumors wereestablished subcutaneously in the nude mice before sera were obtainedfor PA analysis by the injection of cultured cell suspensions. For this,the cells were washed in PBS, counted for viability, and adjusted to thedesired concentration in sterile 0.9 percent NaCl solutions. Subsequentto nodule formation, nude mouse serum was collected by severing theretro-orbital plexus and stored at -20° C. Cell cultures used for theestablishment of human tumor grafts included LNCaP (adenocarcinoma ofthe prostate gland), RT-4 (transitional cell carcinoma of bladder),Palarmo (malignant melanoma), and AsPC-1 (pancreatic carcinoma).

Of the human tumors examined, only the LNCaP prostate line released PAinto the circulation, which correlates with results obtained from theassay of cultured cells. The control mice given preimplants of RT-4cells, Palarmo cells, or AsPC-1 cells showed no detectable PA in theircirculation. It is not presently appreciated if serum antigen levels arecommensurate with the tumor load, a phenomenon previously reported by E.J. Pesce et al. in Cancer Res. 37: 1998-2003 (1977) for the lactatedehydrogenasde enzyme released by human xenografts. If this is so, thenthe human prostate tumor-nude mouse system, coupled with detection ofPA, can provide a valuable clinical model to monitor the effects ofantitumor modalities and the effects of biologic response modifiers forhuman prostate cancer.

EXAMPLE 22 Antiserum Specificity

The optimal concentration of adsorbed antiserum required in therocket-IEP procedure was determined by examining the migration ofprostate antigen in gels containing varying concentrations ofanti-prostate antigen antiserum. The reactivity of various tissues wasexamined by rocket-IEP. As shown in Table 4, all extracts prepared fromnormal prostate, benign hypertrophic and malignant prostate tissuesshowed reactivity, producing a single immunoprecipitin reaction.Extracts prepared from the non-prostate tissues, whether normal orneoplastic in nature, gave no immunologic reactivity.

                  TABLE 4                                                         ______________________________________                                        REACTIVITY OF ANTI-PROSTATE ANTIGEN ANTI-                                     SERUM WITH HUMAN TISSUE EXTRACTS.sup.a                                                          Percent Positive Reaction                                                           Rocket-   Immuno-                                     Tissue    Pathology     IEP       difussion                                   ______________________________________                                        Liver     Normal        0      (0/2)                                                                              0    (0/1)                                Spleen    Normal        0      (0/1)                                                                              0    (0/1)                                Lung      Normal        0      (0/1)                                                                              0    (0/1)                                          Adenocarcinoma                                                                              0      (0/1)                                                                              0    (0/1)                                Bone Marrow                                                                             Normal                    0    (0/1)                                Bladder   Normal        0      (0/1)                                                                              0    (0/1)                                Breast    Normal        0      (0/1)                                                    Adenocarcinoma                                                                              0      (0/5)                                          Intestine Normal        0      (0/1)                                                                              0    (0/1)                                          Adenocarcinoma                                                                              0      (0/1)                                                                              0    (0/1)                                Heart     Normal        0      (0/1)                                                                              0    (0/1)                                Pancreas  Adenocarcinoma                                                                              0      (0/2)                                                                              0    (0/1)                                Kidney    Normal        0      (0/1)                                                                              0    (0/1)                                Cerebral  Normal        0      (0/1)                                                                              0    (0/1)                                Cortex                                                                        Prostate  Normal        100    (4/4)                                                                              100  (20/20)                                        Benign        100    (8/8)                                                                              100  (15/15)                                        Hypertrophic                                                                  Adenocarcinoma                                                                              100    (8/8)                                                                              100  (8/8)                                ______________________________________                                         .sup.a All tissue extracts were adjusted to 10 mg protein/ml prior to         analysis                                                                 

EXAMPLE 23 Diagnostic Specificity

To examine the potential diagnostic value of the prostate antigen, serumsamples were examined for its presence by the method of rocket-IEP,using anti-prostate antigen antiserum treated with normal human plasmato remove antibodies against normal plasma proteins (Table 5). Serumsamples obtained from 20 normal healthy adults and 20 male volunteersover the age of 55 years showed no reactivity against the antiserum withthis assay. Also, serum was drawn from a total of 175 patients withvarious advanced malignancies, including patients with malignancies oflung, colon, rectum, stomach, pancreas, thyroid, breast and withmyeloma. All sera obtained from patients with non-prostatic malignancieswere prostate antigen-negative when assayed by the rocket-IEP procedure.However, out of a total of 219 sera examined from advanced prostaticcancer patients, 17 or approximately 8 percent showed the presence ofprostate antigen in circulation. All sera showing a positive reactionfor prostate antigen were subsequently subjected to the same assay, andreproducibility of the test was 100 percent.

                  TABLE 5                                                         ______________________________________                                        REACTIVITY OF ANTI-PROSTATE ANTIGEN ANTI-                                     SERUM WITH HUMAN SERA BY ROCKET-IEP.sup.a                                     Serum Donors        Percent Positive Reaction                                 ______________________________________                                        Normal adults (male and female)                                                                      0     (0/20)                                           Age-and sex-matched controls                                                                         0     (0/20)                                           Patients with advanced malignancies.sup.b                                     Lung carcinoma         0     (0/83)                                           Thyroid carcinoma      0     (0/1)                                            Colon-rectal carcinoma 0     (0/22)                                           Stomach-pancreas carcinoma                                                                           0     (0/34)                                           Breast carcinoma       0     (0/33)                                           Myeloma                0     (0/2)                                            Prostate carcinoma     8     (17/219)                                         ______________________________________                                         .sup.a All sera were aliquoted and stored at -20° C. or -70.degree     C. until required. A sample volume of 25 μl was used for all studies.      .sup.b Each case was pathologically confirmed.                           

EXAMPLE 24 Enzyme-Linked Immunoassay Tests

Using IgG antibodies against the purified prostate-specific antigen withhorseradish peroxidase and CNBr-activated Sepharose 4B as reagents, asensitive sandwich-type (Sepharose 4B-anti-prostate antigen IgG:prostate antigen: anti-prostate antigen IgG-peroxidase) enzyme-linkedimmunoassay capable of detecting 0.1 ng of prostate antigen/ml. wasevaluated. Of the various normal and tumor tissues examined, only humanprostate tissue was shown to contain the prostate antigen (normalprostate 10±21.9 μg prostate antigen/mg. protein, n=6; benignhypertrophy 18.3±29.5, n=12; malignant prostate 19.1±15.3, n=13).Circulating levels of prostate antigen were also quantitated by the sameassay; no prostate antigen was detectable in sera from normal females(n=17) or female cancer patients (n=25). Additional results (in ng/ml)are shown in Table 6.

                  TABLE 6                                                         ______________________________________                                        ENZYME-LINKED IMMUNOASSAY RESULTS                                             Group      Mean    S.D.   Range n    α 2.30                                                                        p                                  ______________________________________                                        Normal males                                                                             0.47    0.66   0.1-2.6                                                                             51   2                                        Non-prostate                                                                             0.52    0.62   0.1-3.0                                                                             92   3     N.S.                               cancer males                                                                  Prostate cancer                                                               Stage A    8.00    5.64   2.8-14                                                                               3   3     0.02                               Stage B    7.15    3.05   4.6-11                                                                               4   4     0.001                              Stage C    10.52   17.06  0.3-100                                                                             44   31    0.001                              Stage D    22.84   30.97  0.2-270                                                                             250  193   0.001                              ______________________________________                                         (p vs. normal males by Student's t test; N.S. = not significant)         

The prostate antigen from the serum of prostate cancer patients waspurified and shown to be immunochemically identical to the prostateantigen for normal prostate tissue. This data, therefore, demonstratethat the prostate antigen described herein, although ahistotype-specific antigen of the normal prostate, can be used in theimmunological detection of prostatic cancer.

EXAMPLE 25 Prognostic Value of Enzyme-Linked Immunoassay Tests

Using the sensitive enzyme immunoassay reported in Cancer Res. 40:4658(1980), the circulating PA in prostatic cancer patients has beenevaluated clinically. In 96 patients in advanced stage of disease (D₂)and receiving chemotherapies, the pretreatment serum PA levels werefound to be of prognostic value in regard to patients' survival.Patients who survived more than 12 months (n=10) had serum PA levels of11.2 ng/ml±12.7 (mean±S.D.), and those (n=59) who expired within 5months exhibited serum PA values of 28.5±31.9; while levels of 13.7±18.2were found in the patients (n=27) who survived 6-11 months. Nineteen ofthese patients were monitored by 220 serial PA assays for more than 6months, and a clinicopathological correlation between PA levels andclinical course was found in 14 patients (74 percent). Additionally, inanother group of 32 patients who underwent curative therapies forlocalized prostate cancer, 161 serum samples were analyzed during aperiod of 12 to 114 weeks (average 56 weeks). Of these patients, 5developed metastasis during follow-up and all were shown to exhibitincreasingly elevated serum PA values 0-68 weeks preceding the clinicaldiagnosis of disease recurrence. These results demonstrate that PA is auseful new marker for monitoring prostate cancer.

EXAMPLE 26 Establishment of a Hybridoma Cell Line

A nonsecreting myeloma cell line of Balb/c origin described in J.Immunol. 123: 1548-1550 (1979) was used. This line, P3X63Ag8.653, isderived from the γ-1 K-type producing P3X63Ag8. P3X63Ag8.653 cells weremaintained in Dulbecco's modified Eagle's medium (DMEM) containing 15percent heat-inactivated fetal bovine serum (FBS), 2mM glutamine, 100U/ml penicillin, and 100 U/ml streptomycin in a 10 percent CO₂ /airhumidified incubator at 37° C.

Female Balb/c mice (8-10 weeks old) were intraperitoneally injected ondays 1 and 30 with 10 μg Prostate Antigen (PA) purified as described inInvestigative Urology 17:159-163 (1979). Three to six weeks later, themice received an intravenous injection of 10 μg PA in sterile saline.All mice used for fusion experiments showed the presence of serumantibodies against PA, as detected using double immunodiffusion againstpurified PA.

Spleens were aseptically removed from immune mice after the lastimmunization, placed in 10 ml DMEM-15 percent FBS, and cell suspensionsmade by teasing with curved forceps. Clumps and membrane fragments wereallowed to settle and the resulting single cells were washed once bycentrifugation at 600 g for 10 min. Red cells were lysed by incubationin 0.84 percent NH₄ Cl, and the cells were washed and resuspended inDMEM at a concentration of 2×10⁷ cells/ml. The myeloma cells weresimilarly washed and adjusted to 2×10⁷ cells/ml of DMEM. Three ml ofspleen cells were added to 3 ml of myeloma cells and co-pelleted at 600g for 10 min., and the medium was completely removed. For fusion, thecells were gently resuspended in 1 ml of 30 percent (w/v) polyethyleneglycol (PEG), average molecular weight 1,000, in DMEM at pH 8.0. Thetube was rocked for 1 min. and centrifuged for 6 min. at 600 g. After atotal exposure time to the PEG of 8 minutes, the supernatant was removedand 10 ml DMEM slowly added with continuous circular agitation of thetube. Cells were then centrifuged for 10 min. at 600 g, and gentlyresuspended in 50 ml of HAT selection medium (DMEM-15 percent FBScontaining 1.6×10⁻ 5 M thymidine, 1×10⁻⁴ M hypoxanthine, and 1×10⁻⁷ Maminopterin). One ml amounts were dispensed into 48 wells of multiwelldishes and plates were incubated at 37° C. in a humidified atmosphere of10 percent CO₂ in air. HAT medium was replenished every 4 days until day21 when hypoxanthine/thymidine (HT) medium was added. As wells turnedacid (usually 8-12 days after fusion), supernatants were tested forantibody activity. Samples of hybrid cells from wells which showedpositive antibody activity were cloned by limiting dilution and somecells from the same well were transferred to 25 cm² tissue cultureflasks until 2×10⁷ cells were obtained, which were frozen in liquidnitrogen in 10 percent dimethylsulfoxide-90 percent FBS.

EXAMPLE 27 Selection of Hybridoma Cells

Fusion Supernatants were screened for anti-PA antibodies by asolid-phase enzyme immunoassay. Disposable 96-well microtiter plateswere used as the solid adsorbing surface. The wells were filled with 100ml poly-L-lysine succinate (0.25 mg/ml H₂ O) and incubated at 23° C. for15 min. Following 3 washes with PB-NaCl pH 7.2 (50 mM sodiumphosphate:140 mM sodium chloride), 100 ml of purified PA were added at aconcentration of 50 μg/ml 10 mM carbonate buffer, pH 9.6. Afterincubation for 24 hr. at 37° C., 100 ml of 1 percent (w/v) bovine serumalbumin solution in PB-NaCl buffer were added for a further 3 hrs. at37° C.

Prior to performing the antibody screening assay, the microtiter wellswere washed 3 times using PB-NaCl buffer (200 μl). Culture fluids (100μl) to be tested were applied to individual wells and allowed toincubate at 37° C. for 3 hrs., followed by 3 washes with PB-NaCl buffer.Subsequently, 100 μl of peroxidase-conjugated antiserum to mouse Igprepared according to the procedure described in J. Immunol. Methods 15:305-310 (1977) were added to each well and incubated at 37° C. for 3hrs. After 3 washes with PB-NaCl buffer, enzyme activity was revealedusing substrate solution containing 0.08 percent o-dianisidine: 0.003percent H₂ O₂ : 0.01 M sodium phosphate, pH 6.0. The enzyme reaction wasstopped after 60 min. at 23° C. using 25 μl of 1 N HCl.

In each assay, positive control samples were included and consisted ofserially diluted hyperimmune mouse serum raised against purified PA.Tissue culture supernatants from P3X63Ag8 cultures (γ 1-Kappa) and fromthe parental nonsecreting myeloma line were used as negative controls.This screening assay detects immunoglobulins of the IgG, IgM and IgAclasses.

EXAMPLE 28 Cloning of Hybridoma Cells

Desired myeloma hybrids were cloned by limiting dilution in the presenceof peritoneal macrophages to increase cloning efficiency. Hybrid cellswere plated in 96-well culture dishes at a density of 0.5 cells/well inDMEM-15 percent FBS complete medium. Vigorous growth was observed after8-12 days, at which time supernatants were tested from wells showingsingle colonies. Cultures exhibiting antibody activity were re-cloned bythis procedure to ensure population homogeneity.

Peritoneal macrophages were prepared by flushing the peritoneal cavityof Balb/c mice with 5 ml of ice cold, sterile 0.34 M sucrose. To attainhigh yields of macrophages (5-15×10⁶ /mouse), animals received anintraperitoneal injection of 0.5 ml sterile thioglycolate medium 4 daysprior to harvesting the cells. Macrophages were washed once bycentrifugation at 600 g for 5 min. and were resuspended at 10⁶ /ml intissue culture medium. Each tissue culture well received 100 μl ofmacrophage suspension. The hybridoma described herein has been depositedwith the American Type Culture Collection, Rockville, Md. 20850, U.S.A.and is designated ATCC No. HB 8051.

EXAMPLE 29 Purification of Monoclonal Antibodies

Monoclonal antibodies from spent culture fluids were purified usingimmuno-affinity chromatography with rabbit-antimouse Ig: Sepharose 4 Bgel matrix (RaMIg-Sepharose). Twenty ml of R-αMIg:Sepharose were packedinto a chromatographic column (1.5×30 cm) and equilibrated with PB-NaClbuffer. Samples of culture fluids were slowly passed through the column(5 ml/hr), followed by removal of non-specific reactants by elution with100 ml of 0.1 M glycine: 1 M NaCl, pH 9.0. Reactive proteins were theneluted with 3 bed volumes of 4 M KSCN: 0.01 M sodium phosphate, pH 7.2.Dialyzed materials were concentrated using positive-pressureultrafiltration.

EXAMPLE 30 Immunodiffusion Analysis of Secreted Hybridoma Products

An affinity matrix was prepared by coupling 200 mg Ig (R-αMIg) to 5grams of cyanogen bromide-activated Sepharose 4B; an IgG fraction ofrabbit antisera to mouse IgG (H+L) was prepared by a Rivanol proceduredescribed in J. Immunol. Methods 15: 305-310 (1977). Coupling wasperformed at pH 8.0 in bicarbonate buffer according to directionsprovided by the manufacturer (Pharmacia; Piscataway, N.J.). The classand subclass of isolated anti-PA antibodies were determined by doubleimmunodiffusion in 0.6 percent agarose using rabbit anti-mouse IgG1,IgG2a, Ig2b, IgG3, IgM, anti-K and anti-λ chain.

Supernatants from 96 of the most vigorously growing cultures werescreened in duplicate for antibody activity against PA, using a solidphase enzyme-immunoassay. Results indicated that approximately 4 percent(4/96) of cultures were initially antibody-positive. Upon re-assay ofthese cultures, one hybridoma (F5-A-1/22) remained positive. Thisculture was cloned by limiting dilution over peritoneal macrophages andyielded 30 cultures which macroscopically exhibited single colonies ofgrowth. One positive culture was re-cloned and all cultures derivedtherefrom were antibody-positive. These cells (clone F5-A-1/22.8.13,termed Cl. 8.13) were expanded to mass culture and examined for antibodycontent (Table 7). As a positive control for this experiment, culturefluids from P3X63Ag8 cells, known to produce IgG1 kappa-typeimmunoglobulen, were used. Cells of the P3X63Ag8 strain producedapproximately 12 μg/ml of isolated immunoglobulin which was reactiveagainst specific antisera to IGG1 and K-chain (Table 8). Noprecipitation occurred with other immunoglobulen subclass antiserumreagents. Culture fluids from Cl. 8.13 showing anti-PA activitycontained about 10 μg/ml of isolated material using R-αMIg: Sepharosepurification. This isolate was identified as mouse immunoglobulin of theIgM K-type subclass (Table 8). This preparation did not precipitateagainst other subclass of antisera, indicating monoclonality of theisolated immunoglobulin preparation.

                  TABLE 7                                                         ______________________________________                                        Affinity purification of monoclonal antibodies using                          R-αMIg: Sepharose 4B chromatography..sup.a                                        Culture Fluid                                                                 (ml) applied to                                                                              Yield    μgIg/ml                                  Culture   affinity adsorbent                                                                           Ig(mg).sup.b                                                                           culture fluid                               ______________________________________                                        P3X63Ag8  125            1.6      13                                          F5-A-1/22.8.13                                                                          300            3.1      10                                          ______________________________________                                         .sup.a Clarified culture fluids were passed through RαMIg: Sepharos     4B adsorbent as described. The gel was washed with 1M NaCl pH 9.0 until       absorbance at 280 nm reached baseline (less than 0.0200.D.units).             .sup.b RαMIgreactive substances were eluted under chaotropic            dissociating conditions of 4M KSCN. Yield of Ig isolated was determined b     optical density measurements where a 1 percent (w/v) solution of mouse Ig     shows an extinction coefficient of 14.                                   

                  TABLE 8                                                         ______________________________________                                        Immunodiffusion analysis of affinity purified                                 immonoglobulins from spent culture fluids.                                    Affinity isolates from cultures                                                           P3X63Ag8                                                                              F5-a-1/22.8.13                                            ______________________________________                                        R-α IgG1                                                                              +         -                                                     R-α IgG2a                                                                             -         -                                                     R-α IgG2b                                                                             -         -                                                     R-α IgG3                                                                              -         -                                                     R-α K chain                                                                           +         +                                                     R-α λ chain                                                                    -         -                                                     R-α IgM -         +                                                     ______________________________________                                    

The preceding examples can be repeated with similar success bysubstituting the generically or specifically described reactants and/oroperating conditions of this invention for those specifically used inthe examples. From the foregoing description, one skilled in the art towhich this invention pertains can easily ascertain the essentialcharacteristics thereof and, without departing from the spirit and scopeof the present invention, can make various changes and modifications toadapt it to various usages and conditions.

INDUSTRIAL APPLICABILITY

As can be seen from the present specification and examples, the presentinvention is industrially useful in several respects. Although thisprostate antigen is an eutopic product, it appears to be a useful tumormarker such as other differentiated cell products, e.g., prostatic acidphosphatase and thyrocalcitonin. The specificity of prostate antigenantiserum can allow for the identification of neoplastic cells inisolated metastases with unknown primary origin. Anti-prostate antigenantiserum can also be used as a vector to localize and/or carrycytotoxic substances to neoplastic prostatic tissue. Further, the studyof variations in the expression of normal differentiation betweenantigens of prostatic tissue during development of the gland and inprostatic lesions can provide insight into the phenomenon of growthregulation and metastatic ability of prostate cells.

What is claimed is:
 1. An in vivo composition of matter comprising animmunochemically effective concentration and amount of a purified humanprostate specific antigen occurring in normal and cancerous prostatictissue and purified to show a single protein band on analyticalpolyacrylamide gel electrophoresis and isoelectric focusing, saidantigen having a molecular weight as determined by Sephadex G-75 gel offiltration of about 33,000 and by a molecular weight as determined bysodium dodecyl sulfate polyacrylamide gel electrophoresis of about34,000 with no subunit and having an isoelectric point pI of 6.9, saidantigen further being insoluble in perchloric acid and substantiallyfree of normal serum protein components, water-insoluble cellularmaterial and of prostatic acid phosphatases.
 2. An in vitro compositionof matter comprising an immunochemically effective concentration andamount of antibodies against a human prostate specific antigen occurringin normal and cancerous prostatic tissue and purified to show a singleprotein band on analytical polyacrylamide gel electrophoresis andisoelectric focusing, said antigen having a molecular weight by SephadexG-75 gel filtration of about 33,000 and by sodium dodecyl sulfatepolyacrylamide gel electrophoresis of about 34,000 with no subunit andhaving an isoelectric point pI of 6.9, said antigen further beinginsoluble in perchloric acid and said composition being free ofantibodies against human prostatic acid phosphatases.
 3. A compositionaccording to claim 2, wherein said antibodies are monoclonal antibodies.4. A composition according to claim 2, wherein said antibodies areimmunoprecipitating antibodies.
 5. A composition according to claim 2,wherein said antibodies are labeled for radioimmunoassay orenzyme-linked immunoassay.
 6. A composition according to claim 2,wherein said antibodies are covalently bonded to a water-insolublesupport.
 7. A process for preparing immunoprecipitating antibodies toantigens associated with prostatic tissue and fluid, which comprises:(a)extracting an antigen according to claim 1 from prostatic tissue orseparating said antigen from prostatic fluid; (b) separating saidantigen from extraneous antigenic proteins to obtain a compositionconsisting essentially of said antigen; (c) immunizing animals with theresultant purified antigen to form antibodies specific thereto; and (d)recovering immunoprecipitating antibodies against said antigen from saidanimals.
 8. A process according to claim 7, wherein said antigen isseparated from extraneous antigenic protein material by salting out. 9.A process according to claim 7, wherein said antigen is obtained fromseminal fluid.
 10. A process preparing antibodies to antigens associatedwith prostatic tissue and fluid, which comprises:(a) cloning hybridomacells capable of secreting said antibodies; (b) extracting antibodiesaccording to claim 2 from said secretions; (c) separating saidantibodies from extraneous antigenic proteins to obtain a compositionconsisting essentially of said antibodies; and (d) recovering animmunologically effective concentration and amount of said antibodiesfrom said cells.
 11. A process according to claim 10, wherein saidhybridoma cells are prepared by fusing a nonsecretory myeloma cell linewith primary mouse spleen cells.
 12. A method for diagnosing carcinomaof the prostate, which comprises:(a) forming an immunoprecipitin complexbetween (i) an an antigen consisting essentially of the human prostatespecific antigen of claim 1 associated with both normal and cancerousprostatic tissue which is distinct from prostatic acid phosphatase and(ii) immunoprecipitating antibodies thereto which are free ofcross-reactivity against prostatic acid phosphatases and againstantigens associated with other carcinomas; and (b) detecting thepresence of said complex.
 13. A method according to claim 12, whereinthe complex is formed by countercurrent immunoelectrophoresis.
 14. Amethod according to claim 12, wherein the complex is formed byimmunologically reacting said antigen with antibodies thereto covalentlybound to a water-insoluble carrier.
 15. A method according to claim 12,wherein said complex is detected by radioimmunoassay or by enzyme-linkedimmunoassay.
 16. A continuous murine cell line capable of producingmonoclonal antibodies of the IgM isotype to the specific antigen ofclaim 1 under nutrient growth conditions, consisting essentially of afused cell hybrid of:(a) primed antibody-producing cells with (b)myeloma cells capable of producing a homogenous population ofimmunoglobulin in the fused hybridoma.
 17. A cell line according toclaim 16, wherein the fused cell hybrid is ATCC HB 8051.